Purpose　The aim is to develop a method for the analysis of micro-scale amino acids in biological samples.Methods　After having been extracted and dried,the residues were dissolved in a running buffer or a distilled water and acetonitrile.The samples were loaded into column for 5,25 or 50s at a hydrodynamic injection and were separated by capillary electrophoresis.The effect of different dissolvent on the sample stacking was observed.Results　A 100-fold improvement in the amount of material that can be injected into a capillary column without loss of resolution was shown.Conclusion　The marked effect of sample stacking was achieved by the using distilled water and acetonitrile(1∶1,v/v) as dissolvent.

Objective To develop a method for the determination of the dynamic parameters of interaction between methotrexate(MTX)enantiomer and dihydrofolate reductase(DHFR).Methods An affinity capillary electrophoresis(ACE)method was adopted.Using bare fused-silica capillary,the electrophoresis buffer was 50 mmoL/L sodium tetraborate with 0.2%Brij-35,pH 9.50.The temperature of separation was controlled at 25℃ and a voltage of 25 kV was applied.The separation of the reaction mixture was performed at a wavelength of 254 nm.The difference of peak areas about the product was used to calculate the inhibitory rate and IC50 values.Results We establish the detection method for the dynamic parameters of interaction between MTX enantioner and DHFR.The separation of the reaction mixture could be achieved within 30 min.The IC50 value of D-(-)-MTX and L-(+)-MTX were 3.17×10-7 and 2.48×10-8mol/L,respective.The IC50 value of the D-(-)-MTX was 31.67×10-8mol/L,the L-(+)-MTX was2.48×10-8mol/L.The IC50 value of the D-(-)-MTX was about 13 times higher than that of the L-(+)-MTX.Conclusions ACE is a rapid.simple and accurate method that can be used to monitor DHFRdynamic reaction.The IC50 values of MTX enantiomer were quite different.The result first indicated that reaction between MTX enantiomer and DHFR had three-dimensional selection.

Objective To screen a new schistosome vaccine candidate. \ Methods\ Schistosoma japonicum adult cDNA library was screened using sera from immune rabbits vaccinated with irradiated cercariae and monoclonal antibodies against membrane antigen of S.japonicum schistosomula. Three different fragments of S.japonicum cDNA genes were cloned into pGEM-T vector. The sequences of the inserts were determined using an automatic DNA sequencer and were analysed using Blast program. One of the unknown genes (B8) was selected and its ORF sequence (291 bp) was subcloned into eukaryotic expression vector. The recombinant plasmids were identified by restrictive enzymes and PCR amplification. The positive recombinant plasmids (pBK/SjB8) were transformed into host bacteria XL1-blue, and were then induced by IPTG for expression. SDS-PAGE and Western blotting analysis of total cellular protein from the bacteria were performed to detect the gene products. Results The results demonstrated that ORF of SjB8 gene was subcloned into the plasmid pBK-CMV and could express as fusion protein in XL1-blue. The results of SDS-PAGE and Western-blot also showed that the molecular weight of the fusion protein with 3 kDa ?-galactosidase was approximately 13\^6 kDa and the actual molecular weights of the SjB8 was 10\^6 kDa. The expressed fusion product of pBK/Sj-B8 could be recognized by immune serum and McAb. Conclusion A new gene of S.japonicum vaccine candidate (SjB8) was cloned into eukaryotic expression vector pBK-CMV and could express 10\^6 kDa schistosome protein. The results provide foundation for further study of the protein for its posibility as candidate vaccine.

Objective To develop an assay with polyclonal antibodies against a fragment derived from human albumin for determination of a peptide in urine, and to provide an early diagnostic tool for GVHD. Methods A small peptide composed of 16 amino acids (LVRYTKKVPQVSTPTL) was synthesized artificially. The immunogen was then diluted into 100 μg/kg body weight of rabbit. Subcutaneous injection in the immune animals was performed on both sides of spine and groin with 2.5 ml antigen suspension for three times, in order to prepare the polyclonal antibodies. The peptide antigen was then labeled with fluorescein isothiocyanate (FITC), and detected by LIF-CE-IA with the pre-prepared antibodies. Results The titer of serum. The migration time of the labeled antigen was 5.93 min, while the migration time of antigen-antibody complex was 6.47 min. The linear range of the method was 16 to 512 ng/ml, and the minimum detection limit was 10 ng/ml. Conclusions The polyclonal antibodies against the peptide antigen was isolated successfully, which possessed high titer and specificity. These results indicated that the assay was simple and rapid and could be applied for the early diagnosis of patient with GVHD.

OBJECTIVE To investigate the etiology of acute respiratory tract infection(ARI)in Hefei area and risk factors that may influence the distribution of common pathogens.METHODS Direct immunofluorescence assay was applied to detect the respiratory syncytial virus(RSV),adenovirus(ADV),influenza virus A(FluA),influenza virus B(FluB),parainfluenza virus PIV(1,2,3)and real time fluorescent quantitation polymerase chain reaction (FQ-PCR)was applied to measuring Mycoplasma pneumoniae(MP)and Chlamydia pneumoniae(CP)in nasopharyngeal secretions and sputum specimens.RESULTS Among 530 cases included in this study,421 cases (79.43%)showed positive viral etiology.ADV accounted for 73(13.77%),FluA-68(12.83%),FluB-56 (10.56%),RSV-48(9.05%),PIVl-47(8.86%),PIV3-42(7.92%),PIV2-33(6.22%),MP-32(6.03%)and CP-22(4.15%).The detected positive rate of pathogens isolated in winter was the highest(85.07%).CONCLUSIONS Acute upper respiratory tract infection(AURTI)is common and more than 75%pathogens in Hefei area are virus in which the most commonly virus is ADV.Meanwhile atypical pathogens of infection should not be ignored.

Objective To establish a rapid assay for the determination of methotrexate (MTX) in serum.Method The assay was based on the ultraviolet absorbance of methotrexate at 306 nm. The separation of the drug was done by high performance capillary electrophoresis (HPCE). The bare fused-silica capillary was 60 cm in total length, 50.5 cm in efficient length and 75?m in diameter. The voltage of 25 kV was applied. The running buffer was 75 mmol/L phosphate, pH7.4. The performance of methodology was evaluated.Result The complete separation of MTX was achieved within 10 min. The linearity of the assay was from 1.1 ?mol/L to 1100.0 ?mol/L. The minimal detection limit was 0.55 ?mol/L.The recovery of MTX was from 88.2% to 98.2%. Within-run precision was 4.2% and between-run precision was ~5.4%. Conclusion The result indicated that the method was an effective method for clinical and scientific research with advantages of rapidity, simplicity and accuracy.

Objective To explore the feasibility of fluorescence quantitative PCR(FQ-PCR)in prenatal diagnosis of the fetal RhD genotyping using free DNA from RhD-negative pregnant women.Methods The fetal RhD gene was amplified from 78 RhD-negative pregnant women with single fetus maternal plasma (gestation from 11 to 40 weeks).Rhe existence of fetal DNA was confirmed by amplification ofnine different polymorphic short tandem repeat loci(STR)and sex-determining region Y chromosome(SRY)gene.Exon5,7,10 and intron 4 were amplified by real-time polymerase chain reaction with TaqMan probe.The results of fetal RhD genotyping were evaluated retrospectively by the serologic analysis of infant cord blood.Results Among the 78 specimens,the SRY positive signals were detected from samples of 41 and were all identified male fetal through 8ex observation after newborn infants delivered from the women enrolled.The mean concentration of SRY gene reached(214.7±120.9)eopies/ml.RhD genotyping results of 70 cases were in complete concordance with the resets through serological detection of fetal cord blood after delivery.In addition,5 cases were false-positive.3 cages were considered inconclusive.The coincidence rate was 90%(70/78).From 5 false-positive cases,4 cases were identified as RhDel phenotype by detecting RHDl227A allele gene.The final accuracy rate of FQ-PCR was 95%(74/78)in the fetal RhD genotyping.Conclusion FQ-PCR analysis for noninvasive prenatal of fetal RhD genotyping could be useful in prevention and diagnosis of hemolytic disease of newborn.

Objective To investigate the correlation between the expression level of BLM and its clinical significance in leukemia.Methods 125 bone marrow specimens of inpatients and outpatients with leukemia were collected in Anhui provincial hospital from January 2011 to December 2011.125 leukemia patients were diagnosed and classified into acute leukemia (AL,n =66) and chronic myelogenous leukemia (CML,n =59) by Morphologic and Immunologic criteria,5 non-tumor individuals were included as control group.The BLM mRNA expressions were by reverse transcription-polymerase chain reaction(RT-PCR).The specimens were devided into groups according to the age,gender,leukemia type,peripheral blood leukocyte counts,hepatomegalia and(or) splenomegaly,fusion gene,chromosome karyotype,whether first visit and transplantation.The expression of BLM gene in each group and the correlation with above factors were retrospectively analysed.The statistical methods such as chi-square test,single factor variance analysis,t test and Pearson correlation test were mainly used.Results BLM mRNA was detected in leukemia.In bone marrow cells,the BLM gene expression was positive in 71 patients and negative in 54 patients.But none of 5 non-tumor bone marrow cells expressed BLM gene.The difference of BLM expression between patients and controls was statistically significant in two groups,i.e.peripheral blood leukocyte counts and fusion gene (x2 =14.730,22.399 ; P ＜ 0.05),but there is no statistical significant differences in other groups.The expressions of BLM mRNA in leukemia patients who had been treated with chemotherapy were lower than those newly diagnosed (0.1788 ± 0.1091 vs 0.3276 ± 0.2016 ; P ＜ 0.05).Moreover,BLM mRNA level in post-bone marrow transplant patients was lower than those not treated (0.1271 ± 0.1009 vs 0.2902 ±0.2034 ; P ＜ 0.05).Pearson correlation analysis showed that higher BLM mRNA expression positively correlated with fusion gene (r =0.357,P ＜ 0.01) and chromosome abnormality (r =0.279,P ＜ 0.05).Conclusion The BLM mRNA expression level of measurement can be used as judgment for leukemia patients disease severity and the index of prognosis,testing the level may provide a basis for clinical and curative effect judgment.

Objective To establish a rapid and convenient method for determination of genomic DNA methylation in cells.Methods Five standard substances (dC, mdC, dA, dT and dG) were separated by high-performance capillary electrophoresis.Bare fused-silica capillary was used and eletrophoresis buffer was 48 mmol/L NaHCO3 with 60 mmol/L SDS, pH 9.6.The temperature of separation was controlled at 25 ℃ and a voltage of 20 kV was applied.The separation of the mixture was performed at a wavelength of 256 nm with UV-Vis detection and injection time was 5 seconds at 0.7 psi.Under optimal condition,genomic DNA methylation in methotrexate drug-resistant A549 cells was detected.Results The optimal condition was made by adjusting SDS concentration(40, 60, 80 mmol/L), pH value of running buffer(9.4,9.6, 9.8), voltage(15, 17, 19, 20, 22 kV), injection time(5, 10, 15, 20, 30 s) and capillary temperature(15, 20, 25, 30 ℃).The method for determination of genomic DNA methylation in cells was established.Five substances were completely separated by high-performance capillary electrophoresis in 10 mins.Intra-day coefficient of variation was less than 0.2% and inter-day coefficient of variation was less than 2%.The minimal detection limit was 2 μmol/L.Percentage of mdC in A549 parent cells was (4.80 ±0.52) %.Percentage of mdC in 15, 30, 40 μmol/L methotrexate drug-resistant A549 cells were (4.20±0.44) %, ( 3.70 ± 0.36 ) %, (3.10±0.35 ) %, respectively.Conclusions Genomic DNA methylation can be quantificated by high-performance capillary electrophoresis efficiently, rapidly, conveniently and sensitively.Genomic DNA methylation in methotrexate drug resistance cells decreases significantly.

Objective To establish a method for detection folylpolyglutamate syntbetase (FPGS),explore the change of FPGS in the drug-resistant A549 cells induced by methotrexate(MTX) enantiomer,and provide new tools to further investigate drug resistant mechanism. Methods A549 cell lines induced by L-( + )-MTX and D-( - )-MTX (25 μmol/L) were chosen to raise three cell lines as compared with MTX-sensitive cell line. Then FPGS were extracted for the CEIA-LIF and western blot was performed. After validation, FPGS antibodies were labeled by fluoreacein isothiocyanate (FITC) and produced a immune response with former-extracted FPGS. CELA-LIF can separate and detect labeled proteins according ruination time of the protein with different size and detect FPGS in drug resistant cell lines induced with L-(+)-MTX and D-(- )-MTX. The accuracy was evaluated as compared with western blot assay. Results The separation time of CEIA-LIF for labeled FPGS antibody and the immune complexes were7. 1 min and 8.9 min, and the resolving power was 4. 5. The process of protein separation and detection can be accomplished in less than 10 minutes. Western blot analysis showed there was no non-specific bands appears in the extract of these three cell lines after the freeze-thaw in liquid nitrogen. The minimum detection level in sensitive cell strains was 0. 68 mg/μl. The consent of FPGS in L-(+)-MTX and D-( - )-MTX induced cells were 46. 59% and 48. 36% compared with drug sensitive cell strains with CELA-LIF. Conclusions CELA-LIF was established in this experiment. It is efficient and sensitive for detecting of FPGS, which is similar to western blot method. The level of FPGS in L-( + )-MTX and D-( - )-MTX induced drug resistant cell lines is significantly lower, indicating the expression of FPGS is damaged.